Biomineralization plays a crucial role during skeletal and tooth development and allows skeletal tissues to exert their proper functions during adulthood. Despite its obvious importance little is known about its regulation. Regulation of mineral formation in tissues is critical for their proper function. For example, excessive mineral deposition accompanies atheriosclerosis and osteoarthritis. In the latter, crystal formation in the articular surface area may play a major role in the onset of inflammation and the progression of joint destruction. On the other hand, loss of mineralization as seen in osteoporosis leads to fragile bones and bone fractures. Matrix vesicles, small membrane-enclosed particles, have the critical role of initiating mineralization in many tissues, including craniofacial bones, long bones, cartilage and dentin. While it is well established that these particles are released from the plasma membrane, it is unclear how the release of matrix vesicles is regulated and how, following their release into the extracellular matrix, vesicles initiate mineral formation. In our Preliminary Studies it is shown for the first time that only cells undergoing mineralization release annexins II and V-rich, Ca2+/Pi complexes (nucleational core)-containing matrix vesicles which are able to initiate mineralization, while nonmineralizing cells release vesicles which do not mineralize. In addition, we provide evidence that annexins II and V form Ca2+ channels in matrix vesicles, allowing Ca2+ influx into these particles and the formation and growth of the first intralumenal crystals. Release of these annexins II and V-rich matrix vesicles is accompanied by increases in annexins II and V expression and cytosolic Ca2+ concentration, [Ca2+]i. Our hypotheses are that (i) increases in both cytosolic Ca2+ concentration and annexins II and V expression are required for the release of mineralization-competent matrix vesicles, and that (ii) annexins II and V cooperate with the nucleational core to enable Ca2+ influx into the vesicles, formation of the first mineral phase and initiation of mineralization. This project will test these hypotheses directly by the use of diverse experimental strategies, including cell culture, Ca2+ channel studies, site-directed mutagenesis and expression of mutated annexin molecules. The present proposal will provide new insights into some of the most intriguing and important features which regulate mineralization in vivo. This information will be of critical importance for the prevention of uncontrolled mineralization in several tissues during pathological conditions.